Isolated single wood fibres with cellulose fibril angles from 10 to 43° were tested in microtensile tests under controlled temperature and relative humidity of 5, 50, 75, 90% and in the wet state. This systematic study provides experimental stiffness and strength data, calculated on cell wall cross sections. It has been shown that stiffness reduction with increasing moisture content is more pronounced in fibres with large cellulose fibril angles. Interestingly, stiffness reduction in fibres with low cellulose fibril angles has been observed for the fully hydrated state only. The experimental dataset was fed into a model to determine moisture dependent stiffness of the hemicellulose-lignin-matrix and the stresses acting on the fibrils and the matrix.